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Crystal structure and biophysical properties of Bacillus subtilis BdbD. An oxidizing thiol:disulfide oxidoreductase containing a novel metal site.

Crow A, Lewin A, Hecht O, Carlsson Möller M, Moore GR, Hederstedt L, Le Brun NE - J. Biol. Chem. (2009)

Bottom Line: The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR.However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein.Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

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Reduction potential determination for BdbD. A, 1H 15N-HSQC spectra of sBdbD (100 μm) in 50 mm potassium phosphate, pH 7.0, in reduced (red) and oxidized (blue) states. Resonances that were used to follow oxidation state changes are indicated by arrows. The open arrow indicates the resonance shown in more detail in B. The selected reference resonance, which is insensitive to oxidation state, is also indicated (as Ref). B, plot showing potentially dependent changes for one of the selected resonances (reduced resonance at 1H/15N = 8.02/110.54 ppm) at the indicated potential (the reduced resonance is in red and in the oxidized resonance is in blue). As the potential increases the reduced resonance intensity decreases, and intensity corresponding to the oxidized protein (1H/15N = 8.077/110.81 ppm) is observed. C, plot of fraction of reduced sBdbD as a function of the cell potential. The standard deviation for each titration point is indicated on the plot. The solid line shows a fit to supplemental Equation S1. D, thermodynamic cycle connecting the oxidized and reduced forms of native sBdbD with those of unfolded sBdbD (10). K1–K4 are equilibrium constants calculated from ΔG values measured here or, in the case of K3, from literature values for unfolded TDORs/model peptides.
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Figure 6: Reduction potential determination for BdbD. A, 1H 15N-HSQC spectra of sBdbD (100 μm) in 50 mm potassium phosphate, pH 7.0, in reduced (red) and oxidized (blue) states. Resonances that were used to follow oxidation state changes are indicated by arrows. The open arrow indicates the resonance shown in more detail in B. The selected reference resonance, which is insensitive to oxidation state, is also indicated (as Ref). B, plot showing potentially dependent changes for one of the selected resonances (reduced resonance at 1H/15N = 8.02/110.54 ppm) at the indicated potential (the reduced resonance is in red and in the oxidized resonance is in blue). As the potential increases the reduced resonance intensity decreases, and intensity corresponding to the oxidized protein (1H/15N = 8.077/110.81 ppm) is observed. C, plot of fraction of reduced sBdbD as a function of the cell potential. The standard deviation for each titration point is indicated on the plot. The solid line shows a fit to supplemental Equation S1. D, thermodynamic cycle connecting the oxidized and reduced forms of native sBdbD with those of unfolded sBdbD (10). K1–K4 are equilibrium constants calculated from ΔG values measured here or, in the case of K3, from literature values for unfolded TDORs/model peptides.

Mentions: The sensitivity of tryptophan fluorescence intensity to the redox state of TDOR proteins has provided a convenient means to measure their redox potential (23, 36). BdbD lacks the near-active site tryptophan, and so fluorescence methods could not be used to monitor redox state. Therefore, an alternative method was sought. Two-dimensional NMR methods have been used previously to determine the reduction potential of human thioredoxin 1 (64). NMR has the potential to provide much more detailed information about changes occurring in the protein during a redox transition. For example, the effect of redox buffers can readily be monitored through the 1H 15N-HSQC spectrum of a 15N-labeled protein, and the formation of adducts (e.g. mixed disulfides between the protein and glutathione) can be detected. 1H 15N-HSQC spectra of fully oxidized and reduced sBdbD (Fig. 6, A and B) revealed differences in a number of resonances that, although not assigned to specific residues, enabled the redox composition of sBdbD samples to be followed as a function of the potential generated by the GSSG/GSH couple (Fig. 6C). Under the conditions of concentration required for the NMR experiments, the highest potential that could be reliably generated by the GSSG/GSH couple was limited to approximately −80 mV. Normally, this would be more than sufficient to cover the entire transition for a thioredoxin-like protein. In this case, the protein was not fully oxidized at −80 mV, and so a complete redox titration data set was not obtained. Nevertheless, the data could be fitted to the Nernst equation, giving a midpoint reduction potential (Em) of −75 ± 5 mV versus NHE at pH 7 and 25 °C with n, the number of electrons involved in the reduction, at 1.85 ± 0.26. This value of Em is similar to those reported for DsbA from E. coli and S. aureus and is consistent with an oxidative function for BdbD on the outside of the cytoplasmic membrane in B. subtilis.


Crystal structure and biophysical properties of Bacillus subtilis BdbD. An oxidizing thiol:disulfide oxidoreductase containing a novel metal site.

Crow A, Lewin A, Hecht O, Carlsson Möller M, Moore GR, Hederstedt L, Le Brun NE - J. Biol. Chem. (2009)

Reduction potential determination for BdbD. A, 1H 15N-HSQC spectra of sBdbD (100 μm) in 50 mm potassium phosphate, pH 7.0, in reduced (red) and oxidized (blue) states. Resonances that were used to follow oxidation state changes are indicated by arrows. The open arrow indicates the resonance shown in more detail in B. The selected reference resonance, which is insensitive to oxidation state, is also indicated (as Ref). B, plot showing potentially dependent changes for one of the selected resonances (reduced resonance at 1H/15N = 8.02/110.54 ppm) at the indicated potential (the reduced resonance is in red and in the oxidized resonance is in blue). As the potential increases the reduced resonance intensity decreases, and intensity corresponding to the oxidized protein (1H/15N = 8.077/110.81 ppm) is observed. C, plot of fraction of reduced sBdbD as a function of the cell potential. The standard deviation for each titration point is indicated on the plot. The solid line shows a fit to supplemental Equation S1. D, thermodynamic cycle connecting the oxidized and reduced forms of native sBdbD with those of unfolded sBdbD (10). K1–K4 are equilibrium constants calculated from ΔG values measured here or, in the case of K3, from literature values for unfolded TDORs/model peptides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Reduction potential determination for BdbD. A, 1H 15N-HSQC spectra of sBdbD (100 μm) in 50 mm potassium phosphate, pH 7.0, in reduced (red) and oxidized (blue) states. Resonances that were used to follow oxidation state changes are indicated by arrows. The open arrow indicates the resonance shown in more detail in B. The selected reference resonance, which is insensitive to oxidation state, is also indicated (as Ref). B, plot showing potentially dependent changes for one of the selected resonances (reduced resonance at 1H/15N = 8.02/110.54 ppm) at the indicated potential (the reduced resonance is in red and in the oxidized resonance is in blue). As the potential increases the reduced resonance intensity decreases, and intensity corresponding to the oxidized protein (1H/15N = 8.077/110.81 ppm) is observed. C, plot of fraction of reduced sBdbD as a function of the cell potential. The standard deviation for each titration point is indicated on the plot. The solid line shows a fit to supplemental Equation S1. D, thermodynamic cycle connecting the oxidized and reduced forms of native sBdbD with those of unfolded sBdbD (10). K1–K4 are equilibrium constants calculated from ΔG values measured here or, in the case of K3, from literature values for unfolded TDORs/model peptides.
Mentions: The sensitivity of tryptophan fluorescence intensity to the redox state of TDOR proteins has provided a convenient means to measure their redox potential (23, 36). BdbD lacks the near-active site tryptophan, and so fluorescence methods could not be used to monitor redox state. Therefore, an alternative method was sought. Two-dimensional NMR methods have been used previously to determine the reduction potential of human thioredoxin 1 (64). NMR has the potential to provide much more detailed information about changes occurring in the protein during a redox transition. For example, the effect of redox buffers can readily be monitored through the 1H 15N-HSQC spectrum of a 15N-labeled protein, and the formation of adducts (e.g. mixed disulfides between the protein and glutathione) can be detected. 1H 15N-HSQC spectra of fully oxidized and reduced sBdbD (Fig. 6, A and B) revealed differences in a number of resonances that, although not assigned to specific residues, enabled the redox composition of sBdbD samples to be followed as a function of the potential generated by the GSSG/GSH couple (Fig. 6C). Under the conditions of concentration required for the NMR experiments, the highest potential that could be reliably generated by the GSSG/GSH couple was limited to approximately −80 mV. Normally, this would be more than sufficient to cover the entire transition for a thioredoxin-like protein. In this case, the protein was not fully oxidized at −80 mV, and so a complete redox titration data set was not obtained. Nevertheless, the data could be fitted to the Nernst equation, giving a midpoint reduction potential (Em) of −75 ± 5 mV versus NHE at pH 7 and 25 °C with n, the number of electrons involved in the reduction, at 1.85 ± 0.26. This value of Em is similar to those reported for DsbA from E. coli and S. aureus and is consistent with an oxidative function for BdbD on the outside of the cytoplasmic membrane in B. subtilis.

Bottom Line: The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR.However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein.Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

Show MeSH
Related in: MedlinePlus